Construction Of A Recombinant Plasmid Containing Multi-copy CpG Motifs And Its Immuno-modulating Effects On The Immune Responses Of Aquatic Animals, Fish And Prawn

Bacterial DNA and synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG dinucleotides (CpG motifs) regarded as an efficient immunostimulant, have been shown to activate B cells, macrophages, natural killer cells, and dendritic cells directly or indirectly, and induce potential Th1-biased immune responses. However, it is costly to obtain synthetic ODNs, which requires the sacrifice of enormous numbers of aquatic species. In this paper, we reported the construction of a plasmid containing tandem CpG-ODNs (harboring multi-copy CpG motifs), and the evaluation of its immunostimulatory effects on the innate immune responses to aquatic animals in order to obtain and explore a novel anti-pathogen drug in aquaculture.In Part I, multiple CpG-ODNs, previously used effectively in aquatic animals, were designed to be connected one another in series, and inserted quintuple to a plasmid. Then a final recombinant plasmid 'pYES2-90CpG' had been obtained.In Part II, we investigated the immunostimulatory effects of the multiple CpG-ODNs on the immune responses to Crucian carp (Carassius auratus), Japanese seabass (Lateolabrax japonicus), two typical commercial fishes easily infected by pathogens invasions. The results showed that the pYES2-90CpG, compared to the vacant vector, could highly induce the activation of head kidney macrophages and proliferation of peripheral blood leukocytes in Crucian carp and Japanese seabass in vitro. In addition, such plasmid DNA induced the expression of Mx, a I-type IFN induced protein, and upregulated the expression of some important immune-related cytokines including MHC II β, natural killer cell enhancement factor(NKEF) and tumor necrosis factor a(TNF-α) in Crucian carp in vivo. These findings indicated that CpG DNA could be useful tools for understanding the important anti-bacterial defense mechanism in fish.In Part III, we used Pacific white shrimp (Litopenaeus vannamei) as an in vivo model, in turn evaluated the effects of multi-copy CpG motif in plasmid. After 12h of pYES2-90CpG infection, comparative to the pYES2 treatment, we detected the elevation of Lysozyme (LSZ) and Phenoloxidase (PO) activity, and the enhancement of antibacterial activity in prawn haemolymph. It suggested that such multiple tandem CpG-ODNs may be an effective activator on immunity of Pacific white shrimp.It implied that the multi-copy CpG motifs in plasmid could make contribute to these cell- and humoral-mediated immune responses, and might be used not only as an efficient immunoprotectordirectly to cure the diseases of various species but also an adjuvant to enhance the efficacy of vaccine in aquaculture.